Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology Official Journal of the Societa Botanica Italiana ISSN: 1126-3504 (Print) 1724-5575 (Online) Journal homepage: http://www.tandfonline.com/loi/tplb20 Biological activities of Teucrium flavum L., Teucrium fruticans L., and Teucrium siculum rafin crude extracts Rosaria Acquaviva, Carlo Genovese, Andrea Amodeo, Barbara Tomasello, Giuseppe Malfa, Valeria Sorrenti, Gianna Tempera, Alessandro Paolo Addamo, Salvatore Ragusa, Tundis Rosa, Francesco Menichini & Claudia Di Giacomo To cite this article: Rosaria Acquaviva, Carlo Genovese, Andrea Amodeo, Barbara Tomasello, Giuseppe Malfa, Valeria Sorrenti, Gianna Tempera, Alessandro Paolo Addamo, Salvatore Ragusa, Tundis Rosa, Francesco Menichini & Claudia Di Giacomo (2017): Biological activities of Teucrium flavum L., Teucrium fruticans L., and Teucrium siculum rafin crude extracts, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, DOI: 10.1080/11263504.2017.1330773 To link to this article: http://dx.doi.org/10.1080/11263504.2017.1330773 Published online: 02 Jun 2017. Submit your article to this journal Article views: 5 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tplb20 Download by: [5.170.220.45] Date: 05 June 2017, At: 00:17 Plant Biosystems, 2017 https://doi.org/10.1080/11263504.2017.1330773 Biological activities of Teucrium lavum L., Teucrium fruticans L., and Teucrium siculum rain crude extracts Rosaria Acquavivaa, Carlo Genoveseb,c, Andrea Amodeob, Barbara Tomaselloa, Giuseppe Malfaa, Valeria Sorrentia, Gianna Temperab, Alessandro Paolo Addamob, Salvatore Ragusad, Tundis Rosae, Francesco Menichinie and Claudia Di Giacomoa a Biochemistry section, Department of Drug science, University of Catania, Catania, italy; bmicrobiology section, Department of Biomedical and Biotechnological sciences, University of Catania, Catania, italy; cBioBim – interinstitutional multidisciplinary BioBank, iRCCs san Rafaele Pisana, Rome, italy; dDepartment of Health sciences, University “magna Graecia” of Catanzaro, Catanzaro, italy; eDepartment of Pharmacy, Health and nutritional sciences, University of Calabria, Rende, italy ABSTRACT ARTICLE HISTORY The genus Teucrium (Lamiaceae) includes 300 species widespread all around the world, which are perennial herbs or shrubs commonly, named germanders. In Italy, Teucrium lavum L., Teucrium fruticans L., and Teucrium siculum Rain are mostly present in Liguria, Sicily, and Sardegna. Teucrium species are characterized by mono and sesquiterpene hydrocarbons, lavonoids, fatty acid esters, and essential oils. Many species of this genus show antioxidant, antimicrobial, and antifungal activities, rendering them useful as natural preservative ingredients. In view of the interesting biological properties reported for Teucrium spp., in this study we determined the total phenol and lavonoid content of inlorescence extracts of T. lavum L., T. fruticans L., and T. siculum Rain. In addition, we investigated the in vitro antioxidant and antibacterial activities of inlorescence extracts against pathogenic bacteria. Obtained results demonstrated that extracts had in vitro antioxidant activity and showed antimicrobial ability against Gram-positive and Gramnegative strains albeit with diferent efectiveness probably due to the diferent qualitative/quantitative composition of the extract also suggesting that these extracts might be useful in preventing several diseases in which oxidative stress may represent an important pathogenic mechanism. Received 20 July 2016 accepted 9 may 2017 Introduction Since antiquity, many oicinal plants aroused interest as sources of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many infections and preservation of foods from the toxic efects of oxidants. The preservative efect of many plant species and herbs suggests the presence of antioxidative and antimicrobial constituents. Many species, especially those belonging to the Lamiaceae family, such Salvia spp., Origanum spp., and Thymus spp., show strong antioxidant activity. A number of phenolic compounds with strong antioxidant activity have been identiied in these plant extracts (Nakatani 1997). Teucrium spp. (Lamiaceae) is a large genus which includes 300 species distributed in Europe, North Africa, and temperate parts of Asia, but mainly in the Mediterranean region. Teucrium species are perennial herbs or shrubs commonly named germanders (Djabou et al. 2011). Micromorphological characters, especially trichomes, are one of the most useful taxonomic features in Teucrium so that their absence or presence and their typology have a signiicant role in classiication of the genus. CONTACT Rosaria acquaviva © 2017 societá Botanica italiana racquavi@unict.it KEYWORDS Teucrium sp.; oxidative stress; antioxidant capacity; DPPH; soD-like activity; Grampositive; Gram-negative The genus Teucrium is one of the richest sources of diterpenes, with a neoclerodane skeleton: more than 220 diterpenes have been described up to now, and many of these are particularly interesting because of their ecological role as antifeedants against diferent species of insects and for their role in the medicinal properties of the plants (Piozzi et al. 2005). Teucrium species have been used as medicinal plants for more than 2000 years and some of them are still used in folk medicine as antispasmodic, tonic, antipyretic, and antiseptic (Hassan et al. 1979; Velasco Negueruela & Pérez-Alonso 1989). Many Teucrium species are known for their medicinal utilization and exhibit interesting biological properties such as hypoglycemic, hypolipidemic, hepatoprotective, antipyretic, anti-inlammatory, antiulcer, antitumor, antibacterial, and insect antifeedant activities. Teucrium species were used as alimentary plants and some of them are currently used in the preparation of lavored wines, herbal teas, bitters, and liqueurs, as well as leaf and lower infusions are used for lavoring beer in some countries (Maccioni et al. 2007). The importance of this genus and family patterns in food industries lies also on the fact that many species show antimicrobial, antioxidant, and antifungal activities, rendering them useful as natural 2 R. ACQUAVIVA ET AL. preservative ingredients (Özkan et al. 2007; Saroglou et al. 2007; Bezić et al. 2011). Previous phytochemical and pharmacological studies showed that the leaf, lower, and fruit essential oils are characterized by a predominance of sesquiterpenes, such as β-caryophyllene, germacrene D, and β-bisabolene (Lo Presti et al. 2010). T. lavum L., belonging to the Lamiacae family, Sect. Chamaedrys (Miller) Schreber, is an evergreen, branchy, semiwoody shrub, up to 60 cm tall, distributed in the Mediterranean Basin on rocky places. It is characterized by pubescent stems and yellow corolla assembled in terminal spikes. The plant can be found in the cracks of lime rocks from sea level up to 1000 m (Lo Presti et al. 2010; Djabou et al. 2011). It occurs in Italy with two subspecies: the subsp. lavum L. (distributed along the peninsula and in the islands) and the subsp. glaucum (Jord. & Fourr.) L. growing only in Basilicata, Sicily, and Sardinia. In Italian folk medicine, the infusion of the top lowers of this plant was used as antipyretic and antiseptic, whilst the decoction of the leaves was applied directly to the skin as a cicatrizant. T. fruticans L. is a stenomediterranean species particularly present in southern Italy and north Africa that prefers calcareous rocks near the sea. It is widely used as an ornamental plant due to the attractive contrast of its striking blue lowers with its evergreen foliage, which is gray-green above and silver-white beneath. In Italy it grows along the Tyrrhenian coasts up to Naples, in Sicily, Sardinia, and in almost all the minor islands; it is quite rare in the Tremiti Islands and Gargano Promontory. In Southern Tuscany, the leaf infusion is used as depurative and diuretic. Diterpenes and triterpene derivatives have been isolated from T. fruticans L., T. fruticans L. could be classiied among Teucrium species producing germacrene D and β-caryophyllene as the main constituents; however, it also synthesizes high amounts of β-pinene and β-myrcene. Studies of T. fruticans L. have resulted also in the identiication and isolation of other metabolites, including lavonoids that have diverse beneicial biochemical and antioxidant efects (Flamini et al. 2001). T. siculum Rain is an herbaceous perennial shrub, widely spread in central and Southern peninsular Italy, as well as in Sicily (Servettaz et al. 1994), possessing pubescent stems up to 60 cm, growing wild on Mount Etna at altitudes between 900 and 1300 m (Poli Marchese 1991). Several studies reported that extracts of Teucrium spp. extracts exert anti-inlammatory, analgesic, hypotensive, and antioxidant activities (Barrachina et al. 1995; Calatayud et al. 1998). Recently, intense interest has focused on the antioxidant properties of natural products. Several studies have correlated several diseases with oxidative stress and with the lower antioxidant levels (Acquaviva & Iauk 2010; Di Giacomo et al. 2015). Oxidative stress, which results when free-radical formation exceeds protective antioxidant mechanisms or the later are compromised, has become a focus of intense interest in most biomedical disciplines and many types of clinical research; increasing evidence from research show that oxidative stress is associated with inlammation, infections, and cancer and it has been demonstrated that in vitro antibacterial activity can be controlled by redox metabolism suggesting that reactive oxygen species (ROS) are involved in infections and inlammation (Acquaviva et al. 2013; Iauk et al. 2015). In view of the interesting biological properties reported for Teucrium spp. and according with the local traditional medicine, T. lavum L., T. fruticans L., and T. siculum Rain were chosen; these species were also chosen in order to valorize the lora of Mount Etna and considering the geographical location of our University. In the present study inlorescence extracts were used to evaluate in vitro antioxidant activity and antibacterial efects against pathogenic bacteria. Material and methods Chemicals β-Nicotinamide-adenine dinucleotide (NADH), 1,1-diphenyl2-picryl-hydrazyl radical (DPPH), xanthine (X), water, and ethanol used for the extractions were of analytical grade and were purchased from Merck S.p.A. (Milan, Italy); all the other solvent, chemicals, and reference compounds were purchased from Sigma-Aldrich s.r.l. (Milan, Italy). Plant collection and preparation of extracts The inlorescences of T. lavum L., T. fruticans L., T. siculum Rain were collected in Mount Etna (Catania) (Italy) (Lat. N 37°48′25″– 37°44′40″; Long. E 15°4′54″–15°5′21″) in May 2015. The specimens were obtained and authenticated by botanist Prof. S. Ragusa, Department of Health Sciences, University of Catanzaro, Italy. A voucher specimen of the plant (No. 25/03) was deposited in the herbarium of the same Department. Extracts were obtained by maceration, in the dark, of 50 g of air-dried inlorescence in 200 mL of EtOH/H2O (80/20 v/v), for 24 h at room temperature and constantly mixed with a seesaw racker at 20 rpm. The extracts were iltered and evaporated to dryness under reduced pressure with a rotatory evaporator. Thin-layer chromatography (TLC) For the determination of the qualitative chemical proile of the samples, the extracts were analyzed using silica plates (5 × 10 cm, 1 mm height, Merck, Darmstadt, Germany). The mobile phase used was composed of chloroform, methanol, and distilled water (7:13:8, v/v/v) and no spray reagent was used. Total phenol and lavonoid content The concentration of total phenolic compounds was determined spectrophotometrically, using the Folin–Ciocalteau total phenols procedure, as described by Ballard et al. (2010) with modiications. Known amounts of gallic acid were used to prepare the standard curve. Appropriately diluted test extracts (0.1 mL) and the gallic acid standard solutions (0.1 mL) were transferred to 15 mL test tubes. Folin–Ciocalteau reagent (3.0 mL, 0.2 N) was added to each test tube and the contents mixed using a vortex mixer. After 1 min, 9.0% (w/v) Na2CO3 in water (2.0 mL) was added and the solution was mixed. Absorbance was determined at λ = 765 nm. The concentration of total phenolic compounds in the extracts was determined comparing the absorbance between the extract samples and the gallic acid standard solutions. All samples were PLANT BIOSYSTEMS determined in triplicate. Total phenolic content was expressed as μmol gallic acid/l ± standard deviation (SD). The lavonoid content was measured using a colorimetric assay with modiications (Jia et al. 1999). A standard curve of catechin was used for quantiication. Briely ethanolic extracts (25 mL) and/or catechin standard solutions were added to H2O (100 mL). At time zero, 5% NaNO2 (7.5 mL) was added; after 5 min, 10% AlCl3 (7.5 mL) was added, and at 6 min, 1 M NaOH (50 mL) was added. Each reaction mixture was then immediately diluted with H2O (60 mL) and mixed. Absorbances of the mixtures upon the development of pink color were determined as λ = 510 nm. The total lavonoid contents of the samples are expressed as μmol catechin/l. Each result represents the mean ± SD of three experimental determinations. Scavenger efect on superoxide anion (SOD-like activity) Superoxide anion was generated in vitro as described by Acquaviva et al. (2013). A total volume of 1 mL of the assay mixture contained 100 mM triethanolamine–diethanolamine bufer, pH 7.4, 3 mM NADH, 25 mM/12.5 mM EDTA/MnCl2, 10 mM β-mercaptoethanol; samples contained diferent concentrations (0.2–32 μg/mL) of the three extracts of inlorescence of T. lavum L., T. fruticans L., and T. siculum Rain. After 20 min incubation at 25 °C, the decrease in absorbance at λ = 340 nm was measured. Results are expressed as percentage of inhibition of NADH oxidation. SOD (80 mU) was used as reference compound. Each result represents the mean ± SD of ive experimental determinations. Quenching of DPPH The free radical-scavenging capacity of extracts of T. lavum L., T. fruticans L., T. siculum Rain was tested by their ability to bleach the stable DPPH. The reaction mixture contained 86 μM DPPH, diferent concentrations of extracts (10–20–40–80–160 μg/mL) in 1 mL of ethanol. After 10 min at room temperature the absorbance at λ = 517 nm was recorded (Acquaviva et al. 2013). Trolox (30 μM), water-soluble derivative of vitamin E, was used as reference compound. Each result represents the mean ± SD of ive experimental determinations. Antibacterial screening The Minimum Inhibitory Concentrations (MICs) of the three extracts were determined by the broth microdilution method, according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2014). The T. lavum L., T. fruticans L., and T. siculum Rain extracts were previously dissolved in 100% dimethylsulfoxide (DMSO) (10% of total volume) and then in Mueller-Hinton broth (Oxoid) (up to 100% of total volume). The stock concentrations of extracts were 32768 μg/mL. Serial twofold dilutions were made in a concentration range from 16,384 to 8 μg/mL in sterile 96-well plates containing MuellerHinton broth. In total, 32 wild strains of Gram-positive and Gram-negative bacteria isolated from clinical cases were used, including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus 3 mirabilis. The above clinical strains were identiied by standard methods. As control bacteria, S. aureus ATCC 29213, S. epidermidis ATCC 35984, E. faecium ATCC 35667, E. faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 29665, P. mirabilis ATCC 29212 were purchased from the American Type Culture Collection (Rockville, MD). Ten microliter of diluted bacterial suspensions were added to each well to give a inal concentration of 5×105 CFU/mL. The MIC was deined as the lowest concentration at which there was no visible growth after incubation at 37 °C for 24 h. Ciproloxacin was used as antimicrobial positive control. A solvent negative control test was performed to determine the efect of 10% DMSO on the growth of micro-organism. It was observed that 10% DMSO did not inhibit the growth of the micro-organisms. Each test included a positive growth control and a negative sterility control. Results are expressed as mean of three experiments. Statistical analysis One-way analysis of variance followed by Bonferroni’s t test was performed in order to estimate signiicant diferences among samples. Data were reported as mean values ± SD and diferences between groups were considered to be signiicant at p < 0.005. Results Table 1 reports the total phenol and lavonoid contents of the three diferent extracts. T. lavum L. extract resulted richer in phenols and lavonoids compared to T. siculum Rain and T. fruticans L. extracts as conirmed by thin-layer chromatography (TLC) analysis (Figure 1). These extracts inhibited superoxide anion formation in a dose-dependent manner; the T. lavum L. extract has proven the most efective scavenger, with an efect that, at 3.2 μg/mL, was comparable with 80 mU superoxide dismutase (SOD) (Figure 2). The T. siculum Rain and T. fruticans L. extracts exhibited scavenger activities lower than T. lavum L.; the less active was T. fruticans L. extract (Figure 1). The free radical scavenging activity of these extracts was also tested by their ability to bleach the stable DPPH radical (Acquaviva et al. 2013). In this assay, the extracts again showed a DPPH quenching capacity in a dose-dependent manner and T. lavum L. showed a more potent capacity than T. siculum Rain and T. fruticans L. extracts (Figure 3). In addition, at 160 μg/mL, concentration the action of T. lavum L. was equivalent to 30 μM of Trolox (Figure 3). Antimicrobial activity and MIC values exhibited by ethanolic extract of Teucrium species against tested bacterial strains are shown in Table 2. The solvent (10% DMSO) did not inhibit the Table 1. total polyphenols, total lavonoids, and total tannins content in three diferent extracts of T. lavum l., T. siculum Rain, and T. fruticans l. Extract T. flavum l. T. siculum Rain T. fruticans l. Total phenolic content μM gallic acid 90 ± 1 70 ± 2* 53 ± 1* *p < 0.001 vs. T. flavum l. extracts. Total lavonoid content μM catechin 127 ± 2 98 ± 5* 75 ± 3* 4 R. ACQUAVIVA ET AL. E. faecium 028/118 with MIC at 512 μg/mL. For the remaining Grampositive strains, MIC values ranged between 1024 and 8192 μg/mL. The lowest active concentration for six Gram-negative strains was 2048 μg/mL; for the remaining Gram-negative bacteria, MIC values ranged between 4096 and 8192 μg/mL. Teucrium siculum Rain was more active against four Gram-positive strains (S. aureus 004/311, S. epidermidis 042/098, S. epidermidis ATCC 35984, E. faecalis 027/144) with MIC at 256 μg/mL; for the remaining Gram-positive bacteria, MIC values ranged between 512 and 8192 μg/mL. An interesting activity was recorded against Gramnegative bacteria: MIC at 1024 μg/mL against four clinical isolates (K. pneumoniae 004/330, P. mirabilis 036/043, P. mirabilis 042/027, P. mirabilis 019/164); MIC values between 2048 and 8192 μg/mL against the remaining Gram-negative strains. The chemical association of active substances and the phytochemicals concentration can explain the diferent antibacterial properties of Teucrium extracts. Discussion Figure 1. tlC plate for the extracts of T. flavum l., T. siculum Rain, and T. fruticans l. Photograph was digitally enhanced to improve visualization by adjustment of color saturation and brightness/contrast as well as by the eliminate lens distortion. spots (1) T. flavum l; (2) T. siculum Rain; (3) T. fruticans l. growth of micro-organisms. The three extracts showed diferent degree of antimicrobial activity and MIC values ranged from 256 μg/mL to >16,384 μg/mL. T. lavum L. extract was more active against Gram-positive strains, but it showed no remarkable antimicrobial properties against Gram-negative strains. T. fruticans L. and T. siculum Rain extracts showed similar antimicrobial activity and inhibited the growth of all tested bacterial strains, with better activity against Gram-positive micro-organisms. T. lavum L. exhibited better activity against S. epidermidis 053/084, S. epidermidis 042/137 and S. epidermidis 042/098 with MIC value at 1024 μg/mL. K. pneumoniae ATCC 29665 Gram-negative strain was inhibited with MIC value at 8192 μg/mL. T. fruticans L. was more active against S. aureus 004/311, E. faecalis 027/144 with MIC at 256 μg/mL and against S. epidermidis 053/084, S. epidermidis ATCC 35984, E. faecium 014/165, It is known that numerous fruits and vegetables contain high amount of polyphenols which can be beneicial for human health (Yang et al. 2001). Previous studies on ethnomedicine, together with extensive laboratory indings, indicated that lavonoids and monotriterpenic compounds play important roles in the prevention and treatment of several diseases in which oxidative stress is involved (Acquaviva et al. 2016; Bezić et al. 2011; Fung & Brown 2013; Di Giacomo et al. 2015). Prolonged activation of inlammatory cells generates ROS that can damage host DNA and tissues and contribute to infections, diabetes, and carcinogenesis. Polyphenol compounds have received a great deal of attention in recent years due to their powerful antioxidant properties. In T. lavum L., T. fruticans L., T. siculum Rain the content of total phenolic and lavonoid was determined. In particular, T. lavum L. contains greatest quantity of phenolic and lavonoids compounds. These results are in agreement with Djabou et al. who reported that chemical compositions of extracts from subspecies were qualitatively similar but they difered by the normalized % abundances of their major components; in particular, oils from subsp. lavum were dominated by large amounts of hydrocarbon monoterpenes while oils obtained from subsp. fruticans and siculum were characterized by higher amounts of oxygenated compounds (Djabou et al. 2011). Consistent with their diferent polyphenol contents, scavenger activity of the three extracts difered depending on the type of species. In the present study, the ethanol extracts of T. lavum L., T. fruticans L., T. siculum Rain at diferent concentrations were assessed for free radical scavenging and chelating activities in an in vitro model. Since the antioxidant efects of polyphenols could be due both to their free radical scavenging and/or chelating activities, to investigate the superoxide anion scavenging capacity of the extract, we used a method which excludes the Fenton type reaction and the xanthine/xanthine oxidase system. The superoxide anion scavenging activity of the ethanolic extracts of T. lavum L., was higher than that of both T. fruticans L., T. siculum Rain. The free radical scavenging activity of T. flavum L., T. fruticans L., T. siculum Rafin was tested by their ability to PLANT BIOSYSTEMS 5 Figure 2. scavenger efect of extracts of T. flavum l., T. siculum Rain, and T. fruticans l. on superoxide anion; results are expressed as percentage of inhibition of naDH oxidation (rate of superoxide anion production was 4 nmol/min). each value represents the mean ± sD of ive experimental determinations. °p < 0.001 vs. T. flavum l. extracts. Figure 3. scavenger efect of T. flavum l., T. siculum Rain, T. fruticans l., and trolox expressed as capacity to bleach DPPH. Results are expressed as percentage of the decrease in absorbance at λ = 517 nm when compared with the control. each value represents the mean ± sD of of ive experimental determinations. *p < 0.001 vs. diferent concentrations of the same extracts. bleach the stable DPPH radical. This assay provides information on the reactivity of the test sample with a stable free radical. Because of its odd electron, DPPH gives a strong absorption band at 517 nm in visible spectroscopy (deep violet color). As this electron becomes paired off in the presence of a free radical scavenger, the absorption vanishes, and the resulting decolorization is stoichiometric with respect to the number of electrons taken up. In this assay, the T. flavum L., was higher than that of both T. fruticans L., and T. siculum Rafin. The concentrations of the extracts used in the DPPH test are higher than those used to evaluate the scavenger effect on superoxide anion probably due to the larger size and higher stability of DPPH radical respect to O2•The diferent activity of the ethanol extracts could be correlated since ethanol efectively penetrates cellular membranes of plants resulting in the extraction of polyphenols (Sumazian et al. 2010). This suggests that Teucrium species might contain compounds that scavenge and this may account for the regulation of pathological conditions induced by superoxide anion and its oxidation product. The highest amount of phenolics and triterpenic compounds could partially explain why T. lavum L. is more potent than T. fruticans L., T. siculum Rain. In particular, it is 6 R. ACQUAVIVA ET AL. Table 2. antibacterial activity (miC (μg/ml)) of T. lavum l., T. siculum Rain, and T. fruticans l. extracts on Gram-positive and Gram-negative strains. Minimal inhibitory concentrations (μg/mL) Straina Gram-positive strains Staphylococcus aureus 004/311 Staphylococcus aureus 004/162 Staphylococcus aureus 007/172 Staphylococcus aureus 012/089 Staphylococcus aureus atCC 29213 Staphylococcus epidermidis 053/084 Staphylococcus epidermidis 042/137 Staphylococcus epidermidis 042/098 Staphylococcus epidermidis 042/018 Staphylococcus epidermidis atCC 35984 Enterococcus faecium 030/250 Enterococcus faecium 003/102 Enterococcus faecium 014/165 Enterococcus faecium 028/118 Enterococcus faecium atCC 35667 Enterococcus faecalis 029/004 Enterococcus faecalis 027/049 Enterococcus faecalis 027/144 Enterococcus faecalis 021/009 Enterococcus faecalis atCC 29212 Gram-negative strains Escherichia coli 007/070 Escherichia coli 014/065 Escherichia coli 042/023 Escherichia coli 052/118 Escherichia coli atCC 25922 Pseudomonas aeruginosa 027/114 Pseudomonas aeruginosa 001/138 Pseudomonas aeruginosa 018/070 Pseudomonas aeruginosa 018/081 Pseudomonas aeruginosa atCC 27853 Klebsiella pneumoniae 004/330 Klebsiella pneumoniae 036/037 Klebsiella pneumoniae 028/007 Klebsiella pneumoniae 047/020 Klebsiella pneumoniae atCC 29665 Proteus mirabilis 036/043 Proteus mirabilis 042/027 Proteus mirabilis 119/111 Proteus mirabilis 019/164 Proteus mirabilis atCC 29212 Teucrium flavum L. EEb Teucrium fruticans L. EEb Teucrium siculum Rain. EEb DMSOc 10% Cipd 2048 2048 2048 4096 4096 1024 1024 1024 2048 1024 8192 8192 8192 8192 8192 8192 8192 8192 16384 8192 256 1024 2048 2048 2048 512 1024 1024 2048 512 4096 4096 512 512 2048 4096 1024 256 8192 1024 256 512 1024 1024 1024 512 512 256 512 256 1024 4096 512 512 1024 2048 1024 256 8192 1024 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 0,125 0,125 0,25 0,25 0,25 0,032 0,032 0,032 0,064 0,016 1 2 0,5 0,5 1 1 0,5 0,5 2 1 >16384 >16384 >16384 16384 16384 >16384 >16384 >16384 >16384 16384 16384 16384 >16384 >16384 8192 16384 16384 >16384 16384 16384 4096 8192 4096 4096 8192 4096 2048 4096 4096 8192 2048 2048 8192 8192 2048 2048 2048 4096 4096 8192 4096 8192 4096 2048 8192 4096 4096 4096 4096 4096 1024 2048 4096 8192 2048 1024 1024 2048 1024 4096 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 >16384 4 8 4 4 0,5 ≥8 4 ≥8 ≥8 1 4 4 8 8 4 0,016 0,016 1 0,5 0,016 a strain numbers refer to an internal directory for clinical isolates. ee: ethanolic extract. c Dmso: dimethylsulfoxide. d Ciproloxacin. b now recognized that the extremely reactive hydroxyl radical (· OH) derived from O−2 and H2O2 causes DNA strand scission in cellular damage (Halliwell & Gutteridge 1990). Since chronic exposure to free radicals is associated with inlammatory diseases and infections in this study the antibacterial activity of T. lavum L., T. fruticans L., T. siculum Rain was also evaluated. In the last years, the number of bacterial strains resistant to current antibiotics has increased dramatically, thus there is a great need for discovering new antimicrobial agents. Also, the mistrust of antimicrobial agents of synthetic origin due to their potential toxicity and carcinogenicity has intensiied the eforts for discovering new natural bioactive compounds (AnwarMohamed & El-Kadi 2007; Tsay et al. 2007; Theuretzbacher 2011). Concerning the antimicrobial activity, Gram-positive bacteria showed higher sensitivity than Gram-negative bacteria to Teucrium extracts, due to diferences in cell structure: Grampositive bacteria have more mucopeptides in their cell wall composition while Gram-negative bacteria have only a thin layer of mucopeptides and most of their cell structure are lipoproteins and lipo-polysaccharides. Thus, Gram-negative bacteria are more resistant (Tassou & Nychas 1998; Ghalem & Mohamed 2008). The best results in antimicrobial activity were obtained with the extract of T. siculum Rain, with lowest MIC values against both Gram-positive and Gram-negative bacteria. T. fruticans L. demonstrated a good activity against all tested strains. Low antioxidant activity of T. fruticans L. extract justiies its antibacterial activity and becomes crucial in supporting the killer activity of leukocytes which use ROS as a mean against pathogens. Moreover, the concentration of lavonoids and the antimicrobial activity of plant extracts are not directly correlated. This suggests that Teucrium extracts are rich in active compounds not limited to lavonoids, but also a variety of diferent active phenolic compounds. Gursoy and Tepe in 2009 tested polar and non-polar sub-fractions of methanol extracts from T. chamaedrys C. Koch. Whilst polar-subfractions were inactive, non-polar subfractions PLANT BIOSYSTEMS inhibited the growth of Acinetobacter lwoi, C. albicans, C. krusei, Clostridium perfringens, E. coli, and Streptococcus pneumoniae. Bacillus cereus and K. pneumoniae were resistant (Gursoy & Tepe 2009). Antibacterial activity of Teucrium extracts is attributed to the presence of numerous bioactive secondary metabolites. Phenolic compounds, terpenoids, and alkaloids are very important components in antimicrobial activity (Cowan 1999). For isolation of biological components, extraction from plant is one of the more sustainable approaches. The need for selection of most appropriate extraction methodology is evident from the fact that when diferent methods are applied on same plant material with same solvent, extraction eiciency can vary significantly. In addition, the method selected as the most appropriate one also needs to be standardized in order to achieve acceptable degree of reproducibility. It should be noted that choice of appropriate solvent is of essential importance along with application of a compatible extraction method. Some Authors suggest that Teucrium extracts are more active in comparison with acetone and ethyl acetate extracts. The greater efectiveness of the methanol extracts resulted from the fact that methanol is a very useful solvent capable of extracting a wide range of compounds (Ali-Shtayeh & Abu Ghdeib 1999). For the extraction of active components it is most efective to use solvents of high polarity (Stanković et al. 2012). Water–ethanol mixtures are the most suitable solvent systems for the extraction of polyphenols from diferent plants of Lamiaceae family, due to the diferent polarities of the bioactive constituents and the acceptability of this solvent system for human consumption (Akkol et al. 2008; Fecka & Turek 2008; Dent et al. 2013). The results obtained in this study suggest that species belonging to the genus Teucrium represent an important source of pharmacologically active natural products. However further research is still needed for the isolation and puriication of the active compounds and the determination of the mechanisms involved in the antioxidant and antimicrobial activity. Acknowledgments The authors would like to thank Dr M. Wilkinson (Research Assistant) for proofreading the manuscript. 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